Journal: PLoS ONE

Article Title: Metformin Inhibits Growth of Human Glioblastoma Cells and Enhances Therapeutic Response

doi: 10.1371/journal.pone.0123721

Figure Lengend Snippet: ( A ) Four human GB cell lines (U87, U251, LN18 and SF767) were treated with or without metformin (10mM) for 48hrs and Oxygen Consumption Rate (OCR, pmol/min) was measured using Seahorse XF24 Mito stress assay. Data were normalized to total protein content of each well. Metformin treatment significantly reduces OCR (*p<0.05 vs. Control, n = 4). ( B ) Total ATP production and ( C ) glycolytic ATP were determined in metformin-treated or not U87, U251, LN18 and SF767 GB cell lines (*p<0.05, **p<0.01 Met vs. Ctrl, n = 3). ( D ) Lactate concentration (nmol/μL) in U87, U251, LN18 and SF767 cell culture supernatants was determined and shows increased lactate production in response to metformin treatment (*p<0.05, **p<0.01 Met vs. Ctrl, n = 3). (E) Effects of Metformin on rotenone-sensitive ETCI complex activity. (Left panel) GB Cells were treated or not for 48hrs with metformin (Met, 10mM). Mitochondria were isolated and complex I activity was assessed. (Right panel) 10μg of isolated mitochondria from untreated U87 cells were treated for 1hr by rotenone (Rot, 10μM) immediately before the complex I enzymatic assay. Results, expressed as mean ± SEM of at least three independent experiments, represent Complex I relative activities compared to the related control. (**p<0.01, ***p<0.001 compared with the related control).

Article Snippet: Xenograft tumors were generated by injecting either U87 (5x10 5 cells) or LN18 (1x10 6 cells) human glioblastoma cells, in 100μL of PBS, subcutaneously on both flanks of NU/NU athymic mice (n = 5 mice (10 tumors) per group, Charles River).

Techniques: Control, Concentration Assay, Cell Culture, Activity Assay, Isolation, Enzymatic Assay

Journal: PLoS ONE

Article Title: Metformin Inhibits Growth of Human Glioblastoma Cells and Enhances Therapeutic Response

doi: 10.1371/journal.pone.0123721

Figure Lengend Snippet: ( A ) Proliferation assays performed with U87, U251, LN18 and SF767 cells show a decreased cell number in presence of metformin (black curve, Ctrl: PBS vehicle control; grey curve, Met: metformin 10mM) (*p<0.05, **p<0.01, ***p<0.001 Met vs. Ctrl, n = 3). ( B ) Quantification of cell cycle distribution of GB cells, using flow cytometry and PI, staining 48hrs after treatment. Metformin (Met) induces decreased cell number in S phase and increased cell number in G 1 phase compared to control cells (Ctrl). ( C ) Quantification of GB cells in G 0 phase, using flow cytometry and Ki67/PI staining, 12hrs, 24hrs and 48hrs after treatment. Metformin significantly increases the number of cells in G 0 phase (*p<0.05, **p<0.01 Met compared to Ctrl, n = 3).

Article Snippet: Xenograft tumors were generated by injecting either U87 (5x10 5 cells) or LN18 (1x10 6 cells) human glioblastoma cells, in 100μL of PBS, subcutaneously on both flanks of NU/NU athymic mice (n = 5 mice (10 tumors) per group, Charles River).

Techniques: Control, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Metformin Inhibits Growth of Human Glioblastoma Cells and Enhances Therapeutic Response

doi: 10.1371/journal.pone.0123721

Figure Lengend Snippet: ( A ) Quantification of apoptotic and necrotic cell death, using flow cytometry and Annexin-V/PI staining, 12hrs, 24hrs and 48hrs after treatment. Metformin significantly increases the number of AV positive cells (*p<0.05, **p<0.01 compared to control, n = 3). ( B-C ) Quantification of vesicle acidification, using flow cytometry and Acridine Orange staining, following 12hrs, 24hrs or 48hrs treatment in presence or not of 10mM metformin (B) and/or 10μM bafilomycin (C). Metformin increases vesicle acidification for U251, LN18 and SF767 cells. This effect can be reversed using the autophagy inhibitor, bafilomycin (NS: Non-Significant, *p<0.05, **p<0.01, *** p<0.001 n = 5). ( C ) Western Blot analyses for LC3b, Beclin-1 and p62 and respective quantifications showing induction of autophagy in response to metformin treatment (*p<0.05, **p < 0.01 compared to control, n = 3).

Article Snippet: Xenograft tumors were generated by injecting either U87 (5x10 5 cells) or LN18 (1x10 6 cells) human glioblastoma cells, in 100μL of PBS, subcutaneously on both flanks of NU/NU athymic mice (n = 5 mice (10 tumors) per group, Charles River).

Techniques: Flow Cytometry, Staining, Control, Western Blot

Journal: PLoS ONE

Article Title: Metformin Inhibits Growth of Human Glioblastoma Cells and Enhances Therapeutic Response

doi: 10.1371/journal.pone.0123721

Figure Lengend Snippet: ( A ) Western Blot analyses of AMPK and mTOR pathways in U87, U251, LN18 and SF767 cells 48hrs after metformin treatment. Metformin increases AMPK activation leading to increased Acetyl-CoA Carboxylase (ACC) phosphorylation and decreases mTOR/AKT signaling leading to decreased S6K phosphorylation and 4EBP1 hypophosphorylation. Metformin also increases Redd1 expression and decreases HIF-1α expression (*p<0.05, **p < 0.01 compared to control, n = 3).

Article Snippet: Xenograft tumors were generated by injecting either U87 (5x10 5 cells) or LN18 (1x10 6 cells) human glioblastoma cells, in 100μL of PBS, subcutaneously on both flanks of NU/NU athymic mice (n = 5 mice (10 tumors) per group, Charles River).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Expressing, Control

Journal: PLoS ONE

Article Title: Metformin Inhibits Growth of Human Glioblastoma Cells and Enhances Therapeutic Response

doi: 10.1371/journal.pone.0123721

Figure Lengend Snippet: ( A ) Proliferation assays performed with U87, U251, LN18 and SF767 cells show that metformin enhances the anti-proliferative effect of temozolomide (TMZ) (black curve, Ctrl: PBS vehicle control; light grey curve, TMZ: temozolomide 10μM (U87), 5μM (U251), 50μM (LN18), and 30μM (SF767); dark grey curve, TMZ+Met: temozolomide 10μM (U87), 5μM (U251), 50μM (LN18), and 30μM (SF767) and metformin 10mM) (#p<0.05, ##p<0.01, TMZ vs. Ctrl, *p<0.05, **p<0.01, TMZ+Met vs. TMZ, n = 4). ( B ) Quantification of cell death, using flow cytometry and AV/PI staining, 48hrs after metformin treatment and/or TMZ and/or irradiation (IR, 5Gy). TMZ and/or IR combined with metformin present a stronger effect on cell death than TMZ or IR alone, particularly in U87, U251 and SF767 GBM cells (*p<0.05, **p<0.01, n = 4).

Article Snippet: Xenograft tumors were generated by injecting either U87 (5x10 5 cells) or LN18 (1x10 6 cells) human glioblastoma cells, in 100μL of PBS, subcutaneously on both flanks of NU/NU athymic mice (n = 5 mice (10 tumors) per group, Charles River).

Techniques: Control, Flow Cytometry, Staining, Irradiation

Journal: Oncogene

Article Title: Human SLFN5 is a Transcriptional Co-repressor of STAT1-Mediated Interferon Responses and Promotes the Malignant Phenotype in Glioblastoma

doi: 10.1038/onc.2017.205

Figure Lengend Snippet: (A–D) Type-I IFN-dependent expression of human SLFN genes in GBM (A), patient-derived glioma stem cell lines (B), medulloblastoma cell lines (C), and human normal astrocytes SVGp12 (D). Indicated cells were left untreated (UT) or were treated with human IFNα or IFNβ for 6 hours. qRT-PCR analyses of the relative mRNA expression of SLFN5 , SLFN11 , SLFN12 , SLFN13 , and SLFN14 genes are shown. Data are expressed as fold change over untreated controls, and bar graphs represent means ± SEM of three independent experiments for LN18, LN443, SVGp12, and four independent experiments for JK18, JK46, DAOY, D556, LN229 and U87MG. (Und: undetected) (E) Left Panel , Expression of human SLFN proteins in GBM, medulloblastoma cell lines, and normal brain tissue lysates. The cells were lysed and equal amounts of whole cell lysates were resolved by SDS-PAGE. Immunoblots were probed with antibodies against SLFN5, SLFN11, SLFN12L and GAPDH, as indicated. Immunoblot images are representative of five independent experiments for SLFN5, SLFN11 and two independent experiments for SLFN12L. Right panels , bands from five SLFN5, SLFN11, or two SLFN12L independent experiments (including the blots shown) were quantified by densitometry using Image J software and normalized and reported relative to GAPDH.

Article Snippet: LN18, LN229, LN443, U87MG, DAOY and D556 cell lines were subjected to short tandem repeat (STR) analysis to ensure genetic stability and authenticated where published reference STR profiles were available (Genetica DNA Laboratories).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, SDS Page, Western Blot, Software

Journal: Molecular Oncology

Article Title: Allele‐specific proximal promoter hypomethylation of the telomerase reverse transcriptase gene ( TERT ) associates with TERT expression in multiple cancers

doi: 10.1002/1878-0261.12786

Figure Lengend Snippet: Decreased TERT promoter methylation associates with histone marks of active transcription and an active exonic SNP. (A) ChIP‐Bis‐Seq of the TERT promoter using an H3ac antibody shows enrichment of unmethylated DNA in the pulled‐down samples (black) relative to the input (gray) in LN‐18 cells. The absence of any bars indicates zero percent methylation. Inclusion criteria for read positions were a greater number of reads in the pull‐down relative to the input and ≥ 10 reads in the pull‐down (mean input coverage was 9 reads; mean pull‐down coverage was 13 reads; P = 0.01 for pull‐down efficiency). (B) Confirmation of long‐range bisulfite conversion PCR enriching for unmethylated or methylated CpGs at the TERT proximal promoter (16 CpGs spanning 5:1295265–1295396; region overlaps with some of the CpGs analyzed in 3A) using unmethylated (gray)‐ or methylated (black)‐specific bisulfite conversion PCR, respectively. PCR products generated a 1448‐bp product including the proximal promoter and the exon 2 SNP analyzed in Panel C. * P ≤ 0.05 (C) Long‐range bisulfite conversion PCR (same PCRs as shown in Panel B) showing representative Sanger sequencing results (upward arrow indicates position of the exon 2 SNP) and graphs of the sequencing results ( n = 2–3 sequenced reactions). ‘Active SNP’ means that the nucleotide at the position of the SNP is the one found in the TERT mRNA transcribed in that cell line. The active SNP was either previously identified in all cell lines or was identified here (Fig. S6). Error bars represent standard error of the mean. * P ≤ 0.01, where statistical analysis was performed using 2‐tailed Student's t ‐test with unequal variance.

Article Snippet: All media were supplemented with 100 μg·mL −1 penicillin and 100 μg·mL −1 streptomycin (Gibco Thermo Fisher Scientific) and 10% (Sigma‐Aldrich, St. Louis, MO, USA) or 5% (only line LN‐18) fetal bovine serum (FBS) (Peak Serum Inc., Wellington, Colorado, US).

Techniques: Methylation, Generated, Sequencing

Journal: Cancers

Article Title: Downregulation of the Ubiquitin-E3 Ligase RNF123 Promotes Upregulation of the NF-κB1 Target SerpinE1 in Aggressive Glioblastoma Tumors

doi: 10.3390/cancers12051081

Figure Lengend Snippet: Analysis of cell proliferation and invasion in GB cells with RNF123-OV. ( A ) A172, HS683, and LN18 cell lines were stably transfected with empty vector (EV1) or a cDNA encoding Myc- RNF123 (RNF123-OV). RNF123-OV and p50 were assessed by Western blot, and β-actin was used as a loading control. ( B ) Quantification of RNF123 expression by RT-qPCR ( t -test, *** p < 0.001). ( C , D ) Proliferation of LN18 (C) and A172 (D) cell lines with RNF123-OV or the empty vector (EV1) (two-way ANOVA, Bonferroni correction *** p < 0.001). ( E , F ) Colony-forming units LN18 (E) and A172 (F) cell lines stably expressing control empty vector 1 (EV1) or RNF123-OV ( t -test, *** p < 0.001). ( G , H ) Percentage of invasion in LN18 (G) and A172 (H) cell lines stably expressing control (EV1) or RNF123-OV ( t -test, * p < 0.05, ** p < 0.01). ( I ) LN18 cell lines expressing control vector (EV1) or RNF123-OV were analyzed by RNA-sequencing to determine differentially expressed (DE) genes in RNF123-OV cell lines. The image shows a heatmap of the most DE genes (adjusted p < 0.05). ( J ) LN18 (RPPA1) and HS683 (RPPA2) cell lines with RNF123-OV were analyzed by RPPA. The image shows a heatmap of the most DE genes in RNF123-OV cell lines (adjusted p < 0.05). ( K ) Integration of DE genes identified in RPPA1 (LN18), RPPA2 (HS683), and RNA-sequencing in RNF123-OV cell lines that are targets of the NF-κB pathway. ( L ) RT-qPCR for SERPINE1 in A172, HS683, and LN18 cell lines expressing EV1 or RNF123-OV ( t -test, *** p < 0.001). ( M ) Western blot for SerpinE1 in A172, HS683, and LN18 cell lines expressing EV1 or RNF123-OV; low (L) and high (H) exposure times for the same image are shown. ( N ) Correlation analysis of RNF123 and SERPINE1 expression using the TCGA dataset from GB tumors ( n = 145; Spearman’s r = −0.27, p < 0.001). Error bars represent the mean ± SD from n = 3 replicates.

Article Snippet: To establish RNF123 overexpressing clones, LN18, A172, and HS683 cell lines (5 × 10 5 cells in 60 mm dishes) (Corning, NY, USA) were transfected with Myc-tagged RNF123 vector (OriGene, Rockville, MD) using the jetPRIME transfection reagent.

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Western Blot, Control, Expressing, Quantitative RT-PCR, RNA Sequencing

Journal: Cancers

Article Title: Downregulation of the Ubiquitin-E3 Ligase RNF123 Promotes Upregulation of the NF-κB1 Target SerpinE1 in Aggressive Glioblastoma Tumors

doi: 10.3390/cancers12051081

Figure Lengend Snippet: MiR-155-5p decreased RNF123 expression and gave a poor prognosis in GB patients. ( A ) TCGA analysis of a merged cohort of low-grade glioma and GB for copy number variations and mutations. The frequency of RNF123 alteration is 0.8% of a total of 1084 patients. ( B ) Correlation analysis of miR-155-5p and RNF123 expression using TCGA dataset from GB tumors ( n = 145; Pearson r = −0.277, p = 0.0007). ( C ) TCGA database analysis of miR-155 in GB tissue ( n = 202) compared to normal brain tissue ( n = 11) ( t -test, *** p < 0.001). ( D ) Rembrandt database analysis of miR-155 expression in GB tissue ( n = 214) compared to normal brain tissue ( n = 21) ( t -test, *** p < 0.001). ( E ) TCGA database analysis of RNA-sequencing data for miR-155 in IDH WT ( n = 145) or mutated ( n = 8) GB tissue compared to normal brain tissue ( n = 5) (one-way ANOVA, *** p < 0.001, NS = non-significant). ( F ) LN18, A172, and HS683 cell lines were transfected with pre-miR-155-5p (miR-155-5p-OV) or miR control (miR-Ctrl) and RNF123 expression was quantified by Western blot. ( G ) miR-155-5p sequence aligned with human RNF123 WT 3′-UTR (WT) and RNF123 Mutant 3′-UTR (Mut) sequences. ( H ) Luciferase reporter activity assay to determine the effect of miR-155-5p on 3′-UTR of RNF123 using human RNF123 3′-UTR (WT) and RNF123 Mutant 3′-UTR (Mut) sequences cloned in RenSP vector ( t -test, NS = non-significant, *** p < 0.001). ( I ) Percentage of invasion of LN18 cell lines with miR-155-5p-OV, RNF123-OV, or both compared to control cell lines (one-way ANOVA, * p < 0.05, NS = non-significant). ( J ) Correlation analysis of miR-155-5p and SERPINE1 expression using TCGA dataset from GB tumors ( n = 145; Pearson r = 0.368, p < 0.0001). ( K ) GB patients from the TCGA database were split into low ( n = 90) and high ( n = 90) miR-155-5p expression and analyzed for RNF123 expression ( t -test, * p < 0.05). ( L ) Kaplan–Meier curves for the OS of GB patients expressing low ( n = 87) versus high ( n = 87) miR-155-5p (log-rank test, p = 0.024). ( M ) Dot plot to determine miR-155-5p expression in pre-operative plasma from GB patients ( n = 19) and plasma of healthy controls ( n = 46) ( t -test, ** p = 0.024). Error bars represent mean ± SD from replicates ( n = 3).

Article Snippet: To establish RNF123 overexpressing clones, LN18, A172, and HS683 cell lines (5 × 10 5 cells in 60 mm dishes) (Corning, NY, USA) were transfected with Myc-tagged RNF123 vector (OriGene, Rockville, MD) using the jetPRIME transfection reagent.

Techniques: Expressing, RNA Sequencing, Transfection, Control, Western Blot, Sequencing, Mutagenesis, Luciferase, Activity Assay, Clone Assay, Plasmid Preparation, Clinical Proteomics